Eunhee Seol

Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19

Abstract A newly isolated Citrobacter sp. Y19 for CO-dependent H 2 production was studied for its capability of fermentative H 2 production in batch cultivation. When glucose was used as carbon source, the pH of the culture medium significantly decreased as fermentation proceeded and H 2 production was seriously inhibited. The use of fortified phosphate at 60– 180 mM alleviated this inhibition. By increasing culture temperatures (25–36°C), faster cell growth and higher initial H 2 production rates were observed but final H 2 production and yield were almost constant irrespective of temperature. Optimal specific H 2 production activity was observed at 36°C and pH 6–7. The increase of glucose concentration (1– 20 g/l ) in the culture medium resulted in higher H 2 production, but the yield of H 2 production (mol H 2 /mol glucose) gradually decreased with increasing glucose concentration. Carbon mass balance showed that, in addition to cell mass, ethanol, acetate and CO 2 were the major fermentation products and comprised more than 70% of the carbon consumed. The maximal H 2 yield and H 2 production rate were estimated to be 2.49 mol H 2 / mol glucose and 32.3 mmol H 2 / g cell h , respectively. The overall performance of Y19 in fermentative H 2 production is quite similar to that of most H 2 -producing bacteria previously studied, especially to that of Rhodopseudomonas palustris P4, and this indicates that the attempt to find an outstanding bacterial strain for fermentative H 2 production might be very difficult if not impossible.

Fermentative hydrogen production by a new chemoheterotrophic bacterium Rhodopseudomonas Palustris P4

Abstract A newly isolated Rhodopseudomonas palustris P4 for CO-dependent H2 production was studied for its capability of fermentative H2 production in batch cultivations. Important parameters investigated include pH, temperature, concentrations of phosphate and glucose, intermittent purging of culture broth by argon gas, and kind of sugars. The pH of the culture medium significantly decreased as fermentation proceeded due to the accumulation of various organic acids, and this inhibited the H2 production seriously. The use of fortified phosphate at 60– 300 mM could alleviate this inhibition. The increase of glucose concentration (1– 20 g / l ) resulted in higher H2 production, but the yield of H2 production (mmol H2/mmol glucose) gradually decreased with increasing glucose concentration. Intermittent purging of the culture broth by argon gas improved H2 production. Carbon mass balance showed that, in addition to cell mass, ethanol, acetate and CO2 were the major fermentation products that comprised more than 70% of carbon consumed. R. palustris P4 could utilize various monosaccharides (glucose, galactose, fructose), disaccharides (lactose, sucrose) and starch to produce H2. However, the H2 production rate with disaccharides and starch was much slower than that with monosaccharides. The maximal H2 yield and H2 production rate were estimated to be 2.76 mmol H2/mmol glucose and 29.9 mmol H2/g cell h, respectively. These results indicate that, although isolated for CO-dependent H2 production, R. palustris P4 has a high potential as a fermentative H2 producer.

Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H2-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H2 under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H2 production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H2/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H2 producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H2 production is quite similar.

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co‐production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden‐Meyhof‐Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co‐production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner‐Doudoroff (ED) pathway, was greatly enhanced. The maximum co‐production yields were 1.32 mol H2 mol−1 glucose and 1.38 mol ethanol mol−1 glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co‐factors.

Inducible gene expression system by 3-hydroxypropionic acid.

Background3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion.ResultsHere we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP degradation was upregulated by 3-HP by the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory site for LysR binding. Similar inducible systems having an LysR transcriptional regulator were identified in other microorganisms that also could degrade 3-HP. A docking study showed that the 3-HP binding pocket is located between the N-terminal helix-turn-helix motif and the C-terminal cofactor-binding domain.ConclusionsThis LysR-regulated 3-HP-inducible system should prove useful for control of the level of gene expression in response to 3-HP.

Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products

Production of 3-hydroxypropionic acid (3-HP) or 1,3-propanediol (1,3-PDO) production from glycerol is challenging due to the problems associated with cofactor regeneration, coenzyme B12 synthesis, and the instability of pathway enzymes. To address these complications, simultaneous production of 3-HP and 1,3-PDO, instead of individual production of each compound, was attempted. With over-expression of an aldehyde dehydrogenase, recombinant Klebsiella pneumoniae could co-produce 3-HP and 1,3-PDO successfully. However, the production level was unsatisfactory due to excessive accumulation of many by-products, especially acetate. To reduce acetate production, we attempted; (i) reduction of glycerol assimilation through the glycolytic pathway, (ii) increase of glycerol flow towards co-production, and (iii) variation of aeration rate. These efforts were partially beneficial in reducing acetate and improving co-production: 21g/L of 1,3-PDO and 43g/L of 3-HP were obtained. Excessive acetate (>150mM) was still produced at the end of bioreactor runs, and limited co-production efficiency.

Elucidation of toxicity of organic acids inhibiting growth of Escherichia coli W

The toxic effects of 3-hydroxypropionic acid (3-HP) at high concentrations on cell growth and cellular metabolism are a great challenge to its commercial production. This study has examined and compared the toxic effects of 3-HP on cell growth with other similar weak acids, especially lactic acid, under various concentrations, temperatures and pH using Escherichia coli W as the test strain. 3-HP was approximately 4.4-times more toxic than lactic acid due to the 4.4-fold weaker acidity or 0.64 higher pKa value. The two acids presented no appreciable difference when the growth inhibition was correlated with the undissociated or protonated free acid concentration calculated by the Henderson-Hasselbalch equation. The growth inhibition by other small organic acids, such as acetic acid, pyruvic acid, propionic acid, 2-hydroxybutyric acid (2-HB) and 3-hydroxybutyric acid (3-HB), was also well correlated with their pKa values or protonated free acid concentrations. This study suggests that the growth inhibition by small weak acids is mainly caused by the socalled proton effect (rather than the anion effect), i.e., an increase in the intracellular proton concentration. An appropriate increase in the medium pH was suggested to alleviate the acid toxicity by reducing the free acid concentration in the culture medium.

Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

Abstract1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (kcat, 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (Km, mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD+) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag+ and Hg2+. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.

Metabolic engineering of Klebsiella pneumoniae J2B for the production of 1,3-propanediol from glucose

The production of 1,3-propanediol (1,3-PDO) from glucose was investigated using Klebsiella pneumoniae J2B, which converts glycerol to 1,3-PDO and synthesize an essential coenzyme B12. In order to connect the glycolytic pathway with the pathway of 1,3-PDO synthesis from glycerol, i.e., to directly produce diol from glucose, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase from Saccharomyces cerevisiae were overexpressed. Additionally, the effect of expression levels and the use of isoforms of these two enzymes on glycerol and 1,3-PDO production were studied. Furthermore, to prevent loss of produced glycerol, the glycerol oxidation pathways were disrupted. Finally, the conversion rate of glycerol to 1,3-PDO was increased via homologous overexpression of glycerol dehydratase and 1,3-PDO oxidoreductase. The resultant strain successfully produced 1,3-PDO from glucose at a yield of 0.27mol/mol along with glycerol at 0.52mol/mol. Improvement of the engineered K. pneumoniae J2B to further increase conversion of glycerol to 1,3-PDO is discussed.

Deletion of putative oxidoreductases from Klebsiella pneumoniae J2B could reduce 1,3-propanediol during the production of 3-hydroxypropionic acid from glycerol

Recombinant Klebsiella pneumoniae over-expressing 3-hydroxypropionaldehyde (3-HPA) dehydrogenase can produce 3-hydroxypropionic acid (3-HP), an important platform chemical, from glycerol. However, K. pneumoniae co-produces 1,3-propanediol (1,3-PDO) due to the presence of 1,3-propanediol oxidoreductases, which decreases the titer and yield of 3-HP. Previously, two major oxidoreductases, dhaT and yqhD, were removed from K. pneumoniae; however the mutant still produced a significant amount of 1,3-PDO, indicating the probable existence of other oxidoreductase(s). Genome analysis of K. pneumoniae revealed the presence of five putative oxidoreductases having high amino acid similarities to both DhaT (primary 1,3-propanediol oxidoreductase) and YqhD (aldehyde dehydrogenase). Among them, adhE was highly expressed in the absence of DhaT and YqhD. Additionally, an alkyl hydroperoxide oxidoreductase (ahpF), albeit dissimilar to both DhaT and YqhD, was highly expressed in the absence of DhaT and YqhD. To examine the role of adhE and ahpF in 1,3-PDO production, mutant strains devoid of dhaT, yqhD, ahpF and/or adhE genes were developed. However, these mutants neither reduced the production of 1,3-PDO nor improved the production of 3-HP when engineered to over-express an aldehyde dehydrogenase (KGSADH). These results indicate that, apart from DhaT, YqhD, AhpF and AdhE, K. pneumoniae has other, unknown oxidoreductases that are involved in 1,3-PDO production. It is concluded that complete elimination of 1,3-PDO during 3-HP production from glycerol by K. pneumoniae is highly challenging.